The present invention relates to a method for isolating and purifying only a certain unsaturated fatty acid in a high purity from fatty acids present in oils including vegetable oils and fish oils by means of crystallization. More particularly, the present invention relates to a method for isolation and purifying only the desired unsaturated fatty acid in a high purity from fatty acids present in oils by selectively using urea-addition crystallization, and a cooling crystallization or a high liquid chromatography.
Specifically, the present invention provides a method for isolating and purifying linoleic acid or oleic acid as unsaturated fatty acids, in a high purity of at least 99% by subjecting fatty acids derived from oils, particularly, a vegetable oil containing linoleic acid or oleic acid at a high concentration, such as safflower oil, corn germ oil or olive oil, as the raw material to two-step urea-addition crystallization using methanol and urea and then crystallizing the concentrated unsaturated fatty acid from an organic solvent under cooling at temperature of xe2x88x925xc2x0 C. to xcx9cxe2x88x9210xc2x0 C. without stirring
Further, the present invention provides a method for isolating and purifying eicosapentaenoic acid (EPA) as unsaturated fatty acid, in a high purity of at least 99% by subjecting fatty acids derived from oils, particularly, a fish oil containing EPA at a high concentration, such as sardine oil, as the raw material to two-step urea-addition crystallization using methanol and urea to obtain a concentrated unsaturated fatty acid having a high purity and then further purifying the high-purified, concentrated fatty acid by means of a high liquid chromatography using a column filled with Ag-silica or Ag-alumina.
Various animal and vegetable oils, for example, vegetable oils such as safflower oil, corn germ oil and olive oil and fish oils such as sardine oil contain much saturated and unsaturated fatty acids having valuable effects for the food and medicinal purpose. The fatty acids present in such animal and vegetable oils include saturated fatty acids such as palmitic acid, stearic acid, etc., and unsaturated fatty acids such as palmitoleic acid, oleic acid, linoleic acid, linolenic acid, gamm-linolenic acid, arachidonic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), etc.
Among fatty acids derived from such oils, particularly, unsaturated fatty acids have numerous effects useful for food and medicinal purposes and therefore, have been widely used in the field of food and pharmaceutical preparation. The fatty acids present in oils have the following physiological activities. Palmitoleic acid is used as the raw material for cosmetics and a skin protectant; and oleic acid has been known as the raw material for ointments, skin absorbefacient (patch, patch formulation for oral administration, etc.), triolein and synthetic phospholipids, medium for cell culture, etc. Linoleic acid is a source of essential fatty acids and the raw material for cosmetics (vitamin complex) and has an anti-inflammatory activity and an activity for preventing skin cornification; gamma-linolenic acid is a precursor of prostaglandin series 1 and has an effect of improving dermatopathy and an effect of preventing and treating arteriosclerosis and hypertension; and alpha-linolenic acid is a precursor for synthesis of EPA and has an effect of lowering blood cholesterol level and an effect of preventing cardiac disease and adult diseases. EPA has an effect of lowering blood cholesterol and triglyceride levels, inhibiting inflammation and preventing arteriosclerosis and is used as a precursor of prostaglandin series 3. DHA is a fatty acid for constitution of cerebral and ophthalmic cell membrane and has an effect of improving brain function and preventing and alleviating dementia and Alzheimer disease and is used as a precursor of prostaglandin series 3.
However, in order to use such unsaturated fatty acids as the raw material for food and pharmaceutical products they are in need of isolation and purification in a high purity.
For such a purpose numerous methods have been developed. As the method for isolating and purifying unsaturated fatty acids in the prior art, the urea-addition crystallization has been widely known. However, prior urea-addition crystallization could not control the behavior of urea molecular group, and therefore, has been used for the isolation in a mid purity rather than in a high purity. Therefore, when the isolation and purification in a high purity is required particularly for the purpose of medicinal use, there is an urgent need for the development of a novel technique different from the prior urea-addition crystallization technique.
With regard to the prior urea-addition crystallization, the alcoholic -liquid cooling method for simultaneously dissolving fatty acids and urea has been reported in numerous references (e.g. U.S Pat. No.1,240,513; JAOCS, 59,117xcx9c118(March 1982), Haagsma). However, such cooling method could not control the size of urea molecular group, and therefore, has some disadvantages in that urea and urea inclusion compound are simultaneously precipitated in the form of a crystal when the reaction mixture is cooled, and thus, the utility of urea is greatly decreased to the extent that undesirable fatty acids cannot be removed, In order to make up such disadvantages, the necessity for significantly lowering the cooling rate has been raised when the reaction mixture is cooled.
However, the method wherein the cooling rate is lowered as above also has some disadvantages in that the production time is very slow, and further, due to a long stay of unsaturated fatty acids at high temperature the acidification is rapidly proceeded to lower the oxidation stability of fatty acids, so that such method cannot be utilized in a mass-scale production.
Therefore, the necessity for a method for selectively isolating and purifying only the desired unsaturated fatty acid in a high purity from the fatty acid mixture derived from animal and vegetable oils with overcoming the disadvantages involved in the prior methods has been urgently raised. Thus, the present inventors have combined numerous techniques for isolation and purification in a various manner and then assayed the effect of such combined method, As a result, we have identified that the desired unsaturated fatty acids such as linoleic acid, oleic acid or EPA can be isolated in a high purity by conducting the urea-addition crystallization in two steps and then selectively utilizing the cooling crystallization or the high liquid chromatography, as specifically stated below, and then completed the present invention.
That is, in consideration of the fact that by controlling the behavior of the urea molecular group the urea inclusion compound of the desired fatty acids can be perfectly formed even at an high cooling rate without precipitation of urea crystals, the present invention adopts the molecular encapsulation technique, which allows the fatty acids present in the urea inclusion compound to minimally contact with the air, to optionally control the behavior of urea molecular group so that the stability of unsaturated fatty acids can be increased and the selectivity of fatty acids isolation can also be greatly increased to isolate and purify the desired fatty acids in a high purity.
Therefore, the present invention provides a method for isolating and purifying the unsaturated fatty acids very useful for human being, which are a source of energy and further constitute the biological lipids in cell membranes such as vitamins, hormones, etc., by means of a urea-addition crystallization, and then a cooling crystallization or a high liquid chromatography column.
The purpose of the present invention is to provide a method for isolating and purifying unsaturated fatty acids in a high purity of at least 99% by subjecting fatty acids derived from vegetable oils containing linoleic acid or oleic acid at a high concentration or fish oils such as sardine oil containing EPA at a high concentration, as the raw material to two-step urea-addition crystallization or high liquid chromatography.
Another purpose of the present invention is to provide a method for isolating and purifying linoleic acid or oleic acid as unsaturated fatty acids, in a high purity of at least 99% by subjecting fatty acids derived from oils, particularly, a vegetable oil containing linoleic acid or oleic at a high concentration, such as safflower oil, corn germ oil or olive oil, as the raw material to two-step urea-addition crystallization using methanol and urea and then crystallizing the concentrated unsaturated fatty acid from an organic solvent under cooling temperature of xe2x88x925xc2x0 C. to xe2x88x9210xc2x0 C. without stirring.
Still another purpose of the present invention provides a method for isolating and purifying EPA as unsaturated fatty acid, in a high purity of at least 99% by subjecting fatty acids derived from oils, particularly a fish oil containing EPA at a high concentration, such as sardine oil, as the raw material to two-step urea-addition crystallization using methanol and urea to obtain a concentrated unsaturated fatty acid having a high purity and then further purifying the high-purified, concentrated fatty acid by means of a high liquid chromatography using a column filled with Ag-silica or Ag-alumina.